Persistent Reversal of Enhanced Amphetamine Intake by
Transient CaMKII Inhibition
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Citation
Loweth, J. A., D. Li, J. J. Cortright, G. Wilke, O. Jeyifous, R. L.
Neve, K. U. Bayer, and P. Vezina. “Persistent Reversal of
Enhanced Amphetamine Intake by Transient CaMKII Inhibition.”
Journal of Neuroscience 33, no. 4 (January 23, 2013): 14111416.
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http://dx.doi.org/10.1523/jneurosci.4386-13.2013
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Society for Neuroscience
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Final published version
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http://hdl.handle.net/1721.1/79759
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The Journal of Neuroscience, January 23, 2013 • 33(4):1411–1416 • 1411
Brief Communications
Persistent Reversal of Enhanced Amphetamine Intake by
Transient CaMKII Inhibition
Jessica A. Loweth,1,2 Dongdong Li,2 James J. Cortright,2 Georgia Wilke,2 Okunola Jeyifous,3 Rachael L. Neve,4
K. Ulrich Bayer,5 and Paul Vezina1,2
1
Committee on Neurobiology, and Departments of 2Psychiatry and Behavioral Neuroscience and 3Neurobiology, The University of Chicago, Chicago, Illinois
60637, 4Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and 5Department of Pharmacology, University of Colorado Denver,
Aurora, Colorado 80045
Amphetamine exposure transiently increases Ca 2⫹/calmodulin-dependent protein kinase II (CaMKII) ␣ expression in the nucleus
accumbens (NAcc) shell and this persistently increases local GluA1 S831 phosphorylation and enhances behavioral responding to the
drug. Here we assessed whether transiently interfering with CaMKII signaling using a dominant-negative CaMKII␣ mutant delivered to
the NAcc shell with herpes simplex viral vectors could reverse these long-lasting biochemical and behavioral effects observed following
exposure to amphetamine. As expected, transient expression of CaMKII␣ K42M in the NAcc shell produced a corresponding transient
increase in CaMKII␣ and decrease in pCaMKII␣ (T286) protein levels in this site. Remarkably, this transient inhibition of CaMKII activity
produced a long-lasting reversal of the increased GluA1 S831 phosphorylation levels in NAcc shell and persistently blocked the enhanced
locomotor response to and self-administration of amphetamine normally observed in rats previously exposed to the drug. Together,
these results indicate that even transient interference with CaMKII signaling may confer long-lasting benefits in drug-sensitized individuals and point to CaMKII and its downstream pathways as attractive therapeutic targets for the treatment of stimulant addiction.
Introduction
Previous exposure to psychostimulants such as amphetamine
and cocaine enhances subsequent neurochemical and behavioral
responding to the drug. These phenomena are thought to contribute to the transition from casual drug use to addiction
(Robinson and Berridge, 1993; Vezina, 2004). A brain area
prominently linked to the generation of addictive behaviors is the
nucleus accumbens (NAcc) shell (Anderson et al., 2008). We
recently showed that amphetamine exposure transiently increases expression of the Ca 2⫹/calmodulin-dependent protein
kinase II (CaMKII) ␣ isoform in this site and that transient viral
overexpression of CaMKII␣ mimics the behavioral effects of amphetamine exposure in rats (Loweth et al., 2010). Conversely,
NAcc shell infusion of a CaMKII inhibitor (KN93) prevents
cocaine-induced reinstatement of drug seeking (Anderson et al.,
2008), as well as the expression of enhanced NAcc dopamine
overflow (Pierce and Kalivas, 1997), cocaine-induced locomoReceived Sept. 13, 2012; revised Oct. 25, 2012; accepted Nov. 24, 2012.
Author contributions: J.A.L. and P.V. designed research; J.A.L., D.L., J.J.C., G.W., and O.J. performed research;
R.L.N. and K.U.B. contributed unpublished reagents/analytic tools; J.A.L. and P.V. analyzed data; J.A.L., K.U.B., and
P.V. wrote the paper.
This study was supported by grants from the Peter F. McManus Charitable Trust (P.V.), as well as National
Institutes of Health Grants R01 DA09397 (P.V.), R01 NS052644 (K.U.B.), T32 DA07255 (J.J.C. and O.J.), and F31
DA022834 (J.A.L).
The authors declare no competing financial interests.
Correspondence should be addressed to Paul Vezina, Department of Psychiatry and Behavioral Neuroscience, The University of Chicago, 5841 South Maryland Avenue, MC 3077, Chicago, IL 60637. E-mail:
pvezina@yoda.bsd.uchicago.edu. For information regarding CaMKII mutant biochemistry: K. Ulrich Bayer.
E-mail: ulli.bayer@ucdenver.edu.
DOI:10.1523/JNEUROSCI.4386-13.2013
Copyright © 2013 the authors 0270-6474/13/331411-06$15.00/0
tion (Pierce et al., 1998), and amphetamine self-administration
(Loweth et al., 2008), normally observed in sensitized rats. However, KN93 affects not only CaMKII and other protein kinases,
but also L-type Ca 2⫹ channels (Li et al., 1992; Gao et al., 2006),
and an inhibitor of these channels produces the same biochemical (Pierce and Kalivas, 1997) and behavioral effects (Pierce et al.,
1998; Anderson et al., 2008) as KN93. Furthermore, these studies
did not directly assess the possibility that transient NAcc shell
CaMKII inhibition could have long-lasting effects on behavioral
responding to psychostimulants, a possibility suggested by our
earlier work showing that transient increases in CaMKII␣ in this
region lead to long-lasting neuroadaptations that contribute to
the maintenance of sensitization (Loweth et al., 2010). Thus, in
the present study, we used a CaMKII- and brain region-specific
transient inhibition strategy to determine whether interfering directly with endogenous CaMKII signaling within the NAcc shell
could persistently reverse long-lasting behavioral manifestations
of amphetamine sensitization.
Materials and Methods
Strategy for CaMKII inhibition by viral-mediated expression of a K42M
mutant. Replication-deficient herpes simplex virus (HSV) viral vectors
were constructed and packaged as previously described by us (Neve et al.,
1997; Loweth et al., 2010). These vectors were used because they produce
transient overexpression of the transgene that peaks 3– 4 d post-infection
and returns to baseline by day 8 post-infection (Neve et al., 1997; Carlezon and Neve, 2003; Loweth et al., 2010). In addition, local viral infection
permits functional testing of specific groups of neurons in specific brain
areas (here, the NAcc shell). We chose to transiently express a CaMKII␣
K42M mutant that is “kinase dead” due to impaired ATP binding. Such
kinase-dead mutants are commonly used as “dominant-negative” mu-
1412 • J. Neurosci., January 23, 2013 • 33(4):1411–1416
Loweth et al. • CaMKII Inhibition Reverses Enhanced Drug Intake
tants to interfere with endogenous kinase functions, and this has been described for CaMKII
(e.g., Kühl et al., 2000; Xiao et al., 2005; Garic et
al., 2011). Dominant-negative effects can be
exerted by competition with endogenous kinase for upstream activators, downstream substrates, or subcellular targeting modules. In the
case of CaMKII, which forms dodecameric holoenzymes, dominant-negative effects can also be
exerted by the formation of heteromeric holoenzymes, which interferes with the intraholoenzyme inter-subunit autophosphorylation
at T286 that generates Ca 2⫹-independent “autonomous” CaMKII activity (for reviews, see
Coultrap and Bayer, 2012; Lisman et al., 2012). In
addition, the K42M mutant interferes with the
targeting of holoenzymes to the NMDA-type glutamate receptor subunit GluN2 (Bayer et al.,
2006; O’Leary et al., 2011), which is regulated by
nucleotide binding to CaMKII (O’Leary et al.,
2011) and also generates autonomous activity
(Bayer et al., 2001; Bayer et al., 2006). Thus, the
K42M mutant of CaMKII has even greater
dominant-negative potential than the kinasedead mutants of most other kinases. The regulation of CaMKII is rather complex (Coultrap and
Bayer, 2012), but it should be noted that some
cellular functions that simply require Ca 2⫹/
calmodulin-stimulated CaMKII activity may or
may not be affected by the presence of additional
kinase-dead mutant. Thus, the designation as a
dominant-negative mutant (as done here) is always linked to a specific cellular function; for
other functions, a particular kinase-dead mutant
may or may not act in a dominant-negative fashion (for further discussion, see Wayman et al.,
Figure 1. HSV-CaMKII␣ K42M transiently reduces CaMKII␣ T286 autophosphorylation but persistently reverses the increased
2011).
Subjects and surgery. Male Sprague-Dawley phosphorylation of GluA1 at S831 in NAcc shell of amphetamine-exposed rats. a, Rats received NAcc shell microinjections of
(locomotion experiments) and Long–Evans rats HSV-CaMKII␣ K42M or mock infection (INF) 2–3 weeks after exposure to amphetamine (AMPH) or saline. b, c, Regardless of drug
(self-administration experiments) weighing exposure, HSV-CaMKII␣ K42M increased CaMKII␣ protein levels (b) but decreased its T286 autophosphorylation, shown here as a
250 –275 g on arrival were purchased from Har- ratio to total CaMKII␣ (c), on day 4 but not day 8 post-infection. d, The increased phosphorylation of GluA1 at S831 (shown here as
lan Sprague-Dawley and housed individually a ratio to total GluA1) was reversed on days 4 and 8 post-infection with HSV-CaMKII␣ K42M. Representative Western immunoblots
with food and water available ad libitum. They are shown in a. Data are group means ⫾ SEM percentage of saline-mock controls (n ⫽ 4 – 8/group). **p ⬍ 0.01, AMPH-exposed
were implanted with chronic bilateral guide can- mock-infected versus remaining groups; ***p ⬍ 0.001, K42M versus mock; by significant ANOVA followed by LSD tests. e,
nulae as described previously (Loweth et al., Photomicrograph of ␤-galactosidase-positive neurons illustrating the site of infection in NAcc shell (arrow).
2010). For the amphetamine self-administration
studies, rats were also implanted with intraveencodes the protein ␤-galactosidase. LacZ and 10% sucrose vehicle were
nous catheters as described previously (Suto et al., 2004). All surgical proceused interchangeably for mock-infection control infusions as they have condures were conducted using aseptic techniques according to an approved
sistently been found to be without effect (Loweth et al., 2010; Singer et al.,
Institutional Animal Care and Use Committee protocol.
2010). Rats were transported to a biosafety level 2 facility where, as described
Design and procedure. Rats in different groups were exposed to repeated
previously (Loweth et al., 2010; Singer et al., 2010) and according to an
intermittent injections of amphetamine or saline and 2–3 weeks later, inapproved Institutional Biosafety Committee protocol, they were adminisfused intracranially into the NAcc shell with HSV-K42M CaMKII␣ or a
tered bilateral intracranial microinjections into the NAcc shell of their recontrol infusion (mock) consisting of HSV-LacZ or 10% sucrose vehicle.
spective viral vectors or mock control vehicle. Rats were returned to the
Rats in one experiment were tested for their locomotor response to a syscolony room 24 h later.
temic amphetamine injection 4 d and again 8 d following infection. Rats in a
Locomotor testing. To assess the effect of transient expression of
second experiment were tested for their self-administration of amphetamine
CaMKII␣ K42M in NAcc shell neurons on sensitized locomotor rebefore and after HSV infection. Rats in additional groups were killed 4 or 8 d
sponding to amphetamine, rats were assigned randomly to different
following HSV infection to determine the expression pattern of the transgroups based on exposure (systemic amphetamine or saline) and infecgene and assess its effect on phosphorylation of the AMPA receptor GluA1
tion (NAcc shell HSV-K42M CaMKII␣ or mock). Thus, four groups
subunit at S831, a CaMKII residue. In these cases, brain sections were harwere tested: amphetamine-mock, amphetamine-K42M, saline-mock,
vested and subsequently assessed using immunoblotting.
and saline-K42M. Two to 3 weeks after the exposure regimen, rats reExposure injections. Exposure injections (5 ⫻ 1.5 mg/kg amphetamine
ceived their respective NAcc shell microinjections and were tested for
or 1.0 ml/kg saline) were administered every 2–3 d as described previtheir locomotor response to amphetamine (1.0 mg/kg, i.p.) 4 and 8 d
ously (Vezina et al., 2002; Suto et al., 2004). S(⫹)-amphetamine sulfate
later. On each test day, locomotor activity was measured 1 h before and
was obtained from Sigma-Aldrich and dissolved in sterile saline. Doses
2 h after the amphetamine challenge injection using a bank of 12 activity
refer to the weight of the salt.
boxes as described previously (Vezina et al., 2002).
Viral-mediated gene transfer in the NAcc shell. The following HSV vector
Self-administration training and testing. To assess the effects of transient
constructs were used: CaMKII␣ K42M and a control vector, LacZ, which
expression of CaMKII␣ K42M on enhanced amphetamine self-
Loweth et al. • CaMKII Inhibition Reverses Enhanced Drug Intake
J. Neurosci., January 23, 2013 • 33(4):1411–1416 • 1413
Figure 2. Transient NAcc shell CaMKII␣ K42M expression persistently reverses amphetamine-induced locomotor sensitization. a, b, Rats were exposed to amphetamine (AMPH) or saline and, 2–3 weeks
later, received NAcc shell microinjections of HSV-CaMKII␣ K42M or mock-infection (INF) infusions. Locomotor responding to AMPH was assessed when protein levels were elevated (day 4) and once they had
dissipated (day 8). Data are shown to the left as group mean (⫾SEM) 2 h total locomotor counts following the AMPH injection 4 d (a) and 8 d (b) post-infection. In each case, time courses of the locomotor
responsesareillustratedtotherightasgroupmean(⫾SEM)locomotorcountsobtainedbeforeandaftertheAMPHinjection(arrows).TransientexpressionofCaMKII␣ K42MblockedenhancedAMPH-induced
locomotionatbothdays4and8post-infection.**p⬍0.01,***p⬍0.001,AMPH-exposedmock-infectedversusremaininggroups;bysignificantANOVAfollowedbyLSDtests.n⫽6 –7/group.c,Locationof
the injection cannula tips for rats included in the data analyses. Symbols indicate group affiliation. Line drawings are from Paxinos and Watson (2005). Numbers to the right indicate the number of millimeters
from bregma.
administration, separate rats were assigned randomly to one of four groups
based on exposure and infection as described above for locomotor testing.
Two to 3 weeks after the last exposure injection, rats were trained to selfadminister amphetamine on fixed ratio (FR) schedules of reinforcement and
then tested under a progressive ratio (PR) schedule for 4 d pre-infection to
visualize enhanced work output and drug intake in amphetamine-exposed
rats (Vezina et al., 2002). The following day, rats were transported to the
biosafety facility and administered their respective NAcc shell microinjections. PR self-administration testing resumed the next day and continued for
12 d post-infection as described previously (Loweth et al., 2010). Reinforced
lever presses delivered an infusion of amphetamine (200 ␮g/kg/infusion)
through the intravenous catheter. Six rats did not satisfy the FR training
criteria and were excluded from the study. The daily PR test sessions were
terminated after 3 h or after 1 h elapsed without a drug infusion.
Immunoblotting and immunohistochemistry. Brains were removed rapidly
and flash-frozen on dry ice. Sections (1 mm thick) were obtained with a brain
matrix, and 2-mm-diameter punches were taken bilaterally around the injection cannula tips. Tissue punches were processed, loaded (20 ␮g/lane),
run using SDS-PAGE, and developed. The following primary antibodies
were used: CaMKII␣ (1:2000), pCaMKII (T286; 1:1000), GluA1 (1:1000),
pGluA1(S831;1:500), and ␤-actin (1:2000) or tubulin (1:100,000) as a loading control. All antibodies were obtained from Millipore except for ␤-actin
(Sigma-Aldrich). Additional rats were used in immunohistochemistry studies (infection with HSV-LacZ) to visualize the pattern of infection as described previously (Carlezon and Neve, 2003).
Histology. After the completion of the behavioral experiments, rats were
deeply anesthetized and perfused transcardially with 0.9% saline and 10%
formalin, and brains were removed and stained with cresyl violet to identify
rats with injection cannula tips located bilaterally in the NAcc shell. Only rats
with both cannula tips in the targeted region were retained for statistical
analyses, and their number subsequently indicated as n/group in the appropriate figure legends. The number of rats that failed to meet this criterion for
the locomotor and self-administration studies was as follows: amphetaminemock, 7; amphetamine-K42M, 5; saline-mock, 2; saline-K42M, 3.
Data analysis. Immunoblot, locomotor, and self-administration data
were analyzed with two-way between ANOVA with exposure (amphetamine and saline) and infection (HSV-CaMKII␣ K42M and mock) as the
between factors. Post hoc comparisons were made using the least significant
difference (LSD) test. For locomotion, the data analyzed were the total locomotor counts obtained following the amphetamine challenge (2 h) on each
test day. For self-administration, the data analyzed were the total number of
infusions obtained on day ⫺1 pre-infection, on day 4 post-infection (when
expression of the transgene was maximal), and averaged over days 8 –12
post-infection (when the transgene was no longer expressed).
Results
Transient CaMKII inhibition persistently reverses increased
GluA1 S831 phosphorylation in the NAcc shell of
amphetamine-exposed rats
HSV vectors were used to transiently overexpress an inactive mutant
of CaMKII␣ (K42M) in NAcc shell neurons (Fig. 1e) as we have
previously described for active CaMKII␣ (Loweth et al., 2010). Two
to 3 weeks after exposure to amphetamine or saline, rats received
1414 • J. Neurosci., January 23, 2013 • 33(4):1411–1416
Loweth et al. • CaMKII Inhibition Reverses Enhanced Drug Intake
bilateral NAcc shell microinjections of
HSV-CaMKII␣ K42M or mock-infection
control infusions. CaMKII␣ protein and
T286 phosphorylation levels were assessed
on days 4 and 8 post-infection (Fig. 1b,c).
Consistent with previous findings (Loweth
et al., 2010), no changes in either CaMKII␣
protein or T286 phosphorylation levels
were detected 2–3 weeks after exposure to
amphetamine as displayed in the mockinfected rats. Subsequent infection with
HSV-CaMKII␣ K42M transiently increased
total CaMKII␣ protein levels in both
amphetamine- and saline-treated rats; these
returned to baseline levels by day 8 postinfection (Fig. 1b). In contrast, T286 phosphorylation was transiently decreased, both
in absolute terms (data not shown) and as a
ratio of phospho-T286 to total CaMKII␣
(Fig. 1c), confirming that expression of
CaMKII␣ K42M interfered with endogenous CaMKII signaling. This effect was also
transient and no longer evident by day 8
post-infection (Fig. 1c). The ANOVA conducted on CaMKII␣ levels and the ratio
pCaMKII␣/CaMKII␣ showed significant
effects of infection only on day 4 postinfection (F(1,19) ⫽ 20.12, p ⬍ 0.001 and Figure 3. Transient CaMKII inhibition in the NAcc shell of amphetamine-exposed rats persistently reverses enhanced amphetF(1,19) ⫽ 15.90, p ⬍ 0.001, respectively). amine self-administration. Rats were exposed to amphetamine (AMPH) or saline and tested under a progressive ratio schedule of
No other effects, including on day 8 post- reinforcement 2–3 weeks later. a, Group mean ⫾ SEM. AMPH infusions obtained on the days before and after NAcc shell HSVCaMKII␣ K42M or mock infection (INF, arrow). Cumulative presses required to obtain infusions on the progressive ratio schedule
infection, were statistically significant. Thus, are also shown. b, Insets show number of infusions obtained on the day pre-infection (⫺1), day 4 post-infection when the
transient viral expression of CaMKII␣ K42M transgene was maximally expressed, and averaged over days 8 –12 post-infection when the transgene was no longer expressed.
transiently interfered with “autonomy”-in- AMPH-exposed rats obtained significantly more infusions on day ⫺1 pre-infection and CaMKII␣ K42M expression blocked this
ducing CaMKII T286 phosphorylation, enhanced intake on both day 4 and days 8 –12 post-infection. *p ⬍ 0.05, AMPH versus saline exposed; **p ⫽ 0.01, AMPHwhich was back to control levels on day exposed mock-infected versus remaining groups; by significant ANOVA followed by LSD tests. n ⫽ 6 –9/group. c, Location of the
injection cannula tips for rats included in the data analyses as described in Figure 2c.
8 post-infection.
As exposure to a sensitizing amphetamine regimen leads to a long-lasting increase in phosphorylamotor response was completely abolished in the HSV-CaMKII␣
K42M-infected animals, not only on day 4 (Fig. 2a) but also on
tion of GluA1 at S831 in the NAcc shell (Loweth et al., 2010), we
day 8 post-infection (Fig. 2b), again a time point when both
then assessed the effect of transiently inhibiting CaMKII signalCaMKII␣ expression and T286 phosphorylation had returned to
ing in this site on phosphorylation of this GluA1 residue. Again, a
baseline levels (Fig. 1b,c). Significant effects of exposure (day 4,
sustained increase in GluA1 S831 phosphorylation was detected
F(1,21) ⫽ 6.30, p ⬍ 0.05; day 8, F(1,21) ⫽ 4.15, p ⫽ 0.054) and
in the NAcc shell of mock-infected rats 2–3 weeks after exposure
infection (day 4, F(1,21) ⫽ 9.20, p ⬍ 0.01; day 8, F(1,21) ⫽ 4.43, p ⬍
to amphetamine. This increase in phospho-S831 was reversed by
0.05) were detected on both test days. Thus, we next assessed the
CaMKII␣ K42M expression on day 4 (infection, F(1,17) ⫽ 5.70,
p ⬍ 0.05), and, remarkably, remained attenuated on day 8 posteffect of transient CaMKII inhibition on the enhanced amphetinfection (exposure, F(1,18) ⫽ 10.99, p ⬍ 0.01) when both
amine intake observed following amphetamine exposure (Vezina
CaMKII␣ expression and T286 phosphorylation had returned
et al., 2002; Vezina, 2004). Rats were again randomly assigned to
to baseline levels. No other effects were statistically significant.
four groups based on condition of exposure (amphetamine or
These findings were again observed both in absolute terms
saline) and infection (bilateral NAcc shell HSV-CaMKII␣ K42M
or mock infection). As expected (Vezina et al., 2002), rats ex(data not shown) and as a ratio of phospho-S831 to total
posed to amphetamine and given the opportunity to selfGluA1 (Fig. 1d).
administer the drug intravenously worked significantly more and
as a result obtained more infusions compared with salineTransient CaMKII inhibition in the NAcc shell of
exposed controls (Fig. 3; day ⫺1 pre-infection, exposure,
amphetamine-exposed rats persistently reverses their
F(1,28) ⫽ 5.41, p ⬍ 0.05). However, the day following infection
enhanced locomotor response to and self-administration of
through day 4 post-infection when expression of the transgene
the drug
was maximal, amphetamine-exposed rats infected with HSVAs expected, rats exposed to amphetamine 2–3 weeks earlier
CaMKII␣ K42M no longer displayed enhanced amphetamine
showed a greater locomotor response to a systemic amphetamine
intake, obtaining the same number of infusions as saline-exposed
challenge (1.0 mg/kg, i.p.) compared with saline-exposed conrats and significantly fewer than mock-infected amphetaminetrols (Vezina, 2004) (Fig. 2). Remarkably, transient CaMKII inexposed rats (Fig. 3; day 4 post-infection, exposure, F(1,28) ⫽ 3.58,
hibition in the NAcc shell persistently reversed this expression of
p ⫽ 0.07; E ⫻ I, F(1,28) ⫽ 6.69, p ⬍ 0.05). Importantly, this
locomotor sensitization by amphetamine. The enhanced loco-
Loweth et al. • CaMKII Inhibition Reverses Enhanced Drug Intake
reversal of enhanced amphetamine self-administration was
maintained for the full 12 d of testing post-infection and thus
again outlasted the transient interference with CaMKII signaling
achieved by HSV-CaMKII␣ K42M (Fig. 3; days 8 –12 postinfection, E ⫻ I, F(1,25) ⫽ 4.09, p ⫽ 0.05).
Discussion
Expressing a dominant-negative form of CaMKII␣ (K42M) that
transiently inhibits CaMKII activity in NAcc shell neurons persistently blocked the expression of two major manifestations of
sensitization by amphetamine. As expected, previously exposing
rats to amphetamine led to sensitized locomotor responding to a
challenge injection of the drug and, in a separate group of rats,
enhanced drug intake under a PR schedule of reinforcement
(Vezina et al., 2002; Vezina, 2004). Transiently inhibiting CaMKII activity in the NAcc shell of these rats blocked these manifestations of sensitization and continued to do so long after
expression of the transgene had dissipated, indicating that continued uninterrupted CaMKII activation is necessary for their
maintenance. Previous studies suggested that CaMKII activity in
the NAcc shell is required for the expression of stimulant sensitization (Pierce and Kalivas, 1997; Pierce et al., 1998; Loweth et al.,
2008) and cocaine-induced reinstatement (Anderson et al.,
2008). However, these previous studies tested the pharmacological inhibitor KN-93 that is known to affect not only CaMKII and
other protein kinases but also to inhibit L-type Ca 2⫹ channels,
rendering an interpretation of results that focuses specifically on
CaMKII difficult. The use of HSV-mediated delivery of a
CaMKII␣ mutant in the present experiments circumvented these
shortfalls. Importantly, the present study is the first to unequivocally demonstrate that transiently inhibiting CaMKII activity in
the NAcc shell can lead to the persistent and selective reversal of
enhanced behavioral response to amphetamine. Thus, the evidence reported here identifies CaMKII and the signaling pathways it initiates as attractive therapeutic targets for the treatment
of substance abuse.
CaMKII regulates long-term potentiation (LTP) of synaptic
strength, a form of neuronal plasticity thought to underlie both
physiological and addiction-related maladaptive learning and
memory (Loweth et al., 2010; Coultrap and Bayer, 2012). Transient interference with CaMKII signaling has been shown to persistently reverse LTP when it specifically disrupts the CaMKII/
NMDA-type glutamate receptor complex (Buard et al., 2010;
Sanhueza et al., 2011). CaMKII mediates LTP by increasing both
the number and the conductance of synaptic AMPA-type glutamate receptors (Coultrap and Bayer, 2012), the latter by phosphorylating their GluA1 subunit at S831 (Derkach et al., 1999;
Kristensen et al., 2011). As normal LTP and LTP-induced GluA1
S831 phosphorylation require NMDA receptor-bound CaMKII
(Barria and Malinow, 2005; Halt et al., 2012) and the CaMKII␣
K42M mutation disrupts this binding (O’Leary et al., 2011), we
examined the GluA1 S831 phosphorylation state in our animals.
Indeed, previous exposure to amphetamine produced a sustained
increase in GluA1 S831 phosphorylation in the NAcc shell, as
reported previously (Loweth et al., 2010), and it was persistently
reversed by transient CaMKII␣ K42M expression in this site.
Thus, while the interference with CaMKII signaling was transient, its effect on at least one important downstream mediator
persisted, which may have contributed to the persistent reduction in the enhanced behavioral output normally observed in
amphetamine sensitized rats. While GluA1 S831A mutant mice
do not show impairments in hippocampal LTP or LTD (Lee et al.,
2010), they do show deficits in responding for a conditioned
J. Neurosci., January 23, 2013 • 33(4):1411–1416 • 1415
reinforcer (Crombag et al., 2008). Alternatively, an AMPA receptor independent effect of CaMKII␣ on A-type potassium currents was recently proposed to also contribute to behavioral
sensitization by cocaine (Kourrich et al., 2012).
Notably, CaMKII inhibition affected only the enhanced locomotor response and self-administration observed in rats previously exposed to amphetamine. HSV-CaMKII␣ K42M and
mock-infected rats previously exposed to saline showed the same
basal levels of amphetamine-induced locomotion and amphetamine intake throughout testing. These results indicate that continued uninterrupted CaMKII signaling in the NAcc shell is
necessary specifically to maintain enhanced drug intake observed
in individuals that have been repeatedly exposed to the drug but
not for the initial basal intake seen before drug exposure. Although further characterization of the molecular mechanisms
underlying these effects awaits further study, the results reported
here clearly demonstrate that transient inhibition of CaMKII
provides a viable therapeutic strategy to persistently reverse
addiction-related behaviors.
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